PROJECT SUMMARY/ABSTRACT The gut microbiome has been directly implicated in the etiopathogenesis of a number of diseases, including type 2 diabetes (T2D). Over the last decade, human studies have uncovered profound changes in the composition and metabolic function of gastrointestinal microflora in those with metabolic disorders including T2D. However, the mechanism underlying the microbiome's contribution to glucose homeostasis and dysmetabolism remains unclear. Mouse models suggest that one way dysbiosis could contribute to T2D is by altering the bile acid (BA) pool, thus disrupting intestinal metabolic signaling and glucose homeostasis. Bacterial modulation of BA pool could affect host metabolism through two receptors: the farnesoid X receptor (FXR?) and the G protein- coupled BA receptor 1 (TGR5). Both FXR? and TGR5 can modulate GLP-1 signaling from intestinal L-cells in the intestine and colon, further affecting glucose homeostasis. However, there are few tools available for researchers to study the functional relationship of the gut microbiome, BA signaling, and host metabolism. As a result, the relationships between specific microbiota, bacterial pathways that metabolize BAs, modulations of the BA pool, their effect on incretin signaling, and host glucose homeostasis are poorly understood. A potential method to understand the functional role of the microbiome is to use engineered probiotics (EnProbs), commensal bacteria that can colonize the gut of the host and have a genome that can be easily manipulated. The primary hypothesis of this proposal is that manipulation of the luminal BA pool with conjugating and de-conjugating EnProbs will affect gut incretin response, hepatic gluconeogenesis, and overall host glucose homeostasis. The proposal describes two specific aims to be done in two years that investigate the functional role of the gut microbiome in BA signaling using EnProbs. In the first specific aim, the effects of two different BA modifying EnProbs, a BA conjugating EnProb and a BA deconjugating EnProb, are on the luminal BA profile, intestinal incretin release through TGR5 BA signaling, and insulin sensitivity. In the second specific aim, the effects of two different BA modifying EnProbs on intestinal FXR? BA signaling by measuring the expression of its downstream target genes in the intestine, serum BA profile, and gluconeogenesis in hepatocytes and skeletal myocytes, as well as overall host glucose homeostasis. These comprehensive analyses will produce a better understanding of the functional role of a specific bacterial pathway in BA signaling and overall host metabolism and help develop EnProbs as a novel tool for functional analysis of the gut microbiome. Ultimately, these experiments will help identify novel therapeutic targets, and may perhaps show that EnProbs themselves can be used as therapeutic agents.